Pyrrolobenzodiazepines (PBDs) are a group of naturally occurring compounds that were discovered in the cultures of Streptomyces in the 1960s. Some natural PBDs discovered in these cultures, such as anthramycin and sibiromycin, were shown to possess a broad spectrum of anti-tumor activity. Since cancer is still a leading cause of death globally, the development of novel anti-proliferative derivatives of PBDs is essential for human welfare worldwide. Further synthesis and structure-activity relationship (SAR) studies of the parent natural products and their tetracyclic analogs will lead to the discovery of drug candidates. In this work, thirteen PBD analogues were synthesized using no more than three to four synthetic steps, beginning with commercially obtainable L-proline and isatoic anhydride. The MTT assay, which is a colorimetric assay that uses 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) to assess cell metabolic activity, was initially implimented to test the in vitro cytotoxicity of the compounds using multiple cell lines, namely: SKBR-3, MCF-7, SKMEL-2, CaCo 2, HCT 116, and Mia Paca. Nearly all of the compounds decreased the cell viability of MCF-7 by roughly 20%. Additionally, the anti-proliferative activity of the PBD products were further evaluated by the NCI-60 Human Tumor Cell Lines Screen, which is a part of the National Cancer Institute’s Development Therapeutics Program - Drug Synthesis and Chemistry Branch.